Polymer particles for neutrophil injury

ABSTRACT

Provided herein are methods of treatment, compositions, systems and kits using polymer particles as restraints of neutrophil function. Such methods include, but are not limited to, methods of preventing, treating, and/or ameliorating inflammatory diseases, infections, autoimmune diseases, malignant diseases, and other diseases or conditions in which neutrophils may be implicated. In some embodiments, polymer particles are useful for diagnosing neutrophil related diseases or conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationSer. No. 62/870,879 filed Jul. 5, 2019, the entirety of which isincorporated by reference herein.

FIELD OF THE INVENTION

Provided herein are methods of treatment, compositions, systems and kitsusing polymer particles as restraints of neutrophil function. Suchmethods include, but are not limited to, methods of preventing,treating, and/or ameliorating inflammatory diseases, infections,autoimmune diseases, malignant diseases, and other diseases orconditions in which neutrophils may be implicated. In some embodiments,polymer particles are useful for diagnosing neutrophil related diseasesor conditions.

BACKGROUND OF THE INVENTION

Neutrophil-based medical conditions comprise a diversity of medicalconditions including vascular thrombosis, inflammatory arthritides,systemic lupus erythematosus (SLE), atherosclerosis, sepsis and acutelung injury. For example, acute lung injury (ALI) is a rapidlyprogressing inflammatory disease characterized by disruption of the lungendothelial and epithelial barriers leading to accumulation of fluids inthe alveolar airspace. Blood-gas barrier damage impairs gas exchange andreduces lung function. ALI together with acute respiratory distresssyndrome (ARDS), a more severe form of ALI, affects 200,000 patients peryear in the US, with a mortality rate of ˜40% with a mortality rate of˜50-60% up to 6 months after hospital discharge. No pharmacologicalintervention is effective in reducing mortality in ALI/ARDS. Forexample, nitric oxide to decrease ARDS-related pulmonary hypertension,exogenous surfactants, intravenous prostaglandin E1, and glucocorticoidshave shown no benefit in resolving ALI/ARDS. The primary treatment forARDS is supportive with use of a mechanical ventilator for bloodoxygenation and CO2 removal, thereby allowing the damaged lung to heal.However, further damage to the lung may occur with mechanicalventilation if not employed with care. Hence, management of ALI/ARDS isan unmet clinical need.

SUMMARY OF THE INVENTION

Provided herein are methods of treatment, compositions, systems and kitsusing polymer particles as restraints of neutrophil function. Suchmethods include, but are not limited to, methods of preventing,treating, and/or ameliorating inflammatory diseases, infections,autoimmune diseases, malignant diseases, and other diseases orconditions in which neutrophils may be implicated. In some embodiments,polymer particles are useful for diagnosing neutrophil related diseasesor conditions.

In some embodiments, provided herein are methods of treating,ameliorating, or preventing recurrence of a neutrophil-mediatedinflammatory condition in a patient comprising administering to thepatient a therapeutically effective amount of a salicylate polyanhydrideester that hydrolyzes to salicylic acid (as used herein “Poly-A”)particle and pharmaceutically acceptable carrier, and/orpharmaceutically acceptable formulation. In certain embodiments, thePoly-A particle is a vascular-targeted particle (VTP). In otherembodiments, the Poly-A particle is a non-targeted particle (nTP). Inparticular embodiments, the neutrophil-mediated condition is one or moreconditions selected from vascular thrombosis, inflammatory arthritis,systemic lupus erythematosus (SLE), atherosclerosis, sepsis, arthritis,acute lung injury (ALI) and acute respiratory distress syndrome (ARDS).In particular embodiments, the patient is a mammal. In otherembodiments, the mammal is a human.

In some embodiments, the administering is intravascular administering.In certain embodiments, the administering is intravenous administering.In further embodiments, the administering is administering using aguided catheter guided by, for example, X-radiology imaging, ultrasoundimaging, computerized axial tomography imaging, and/or magneticresonance imaging.

In some embodiments, the Poly-A particle is a microparticle with adimension, for sample, of 500 to 900 nm or greater. In otherembodiments, the Poly-A particle is a nanoparticle with a dimension, forexample, of less than 500 nm. In certain embodiments, the Poly-Aparticle is a sphere. In other embodiments, the microparticle is amicrosphere. In particular embodiments, the sphere comprises a smoothsurface. In further embodiments, the Poly-A particle comprises adiversity of Poly-A particles that differ in dimension, shape, and/orsurface morphology.

In some embodiments, the present invention provides a kit comprising apharmaceutical composition comprising a Poly-A particle, and,optionally, instructions for administering the pharmaceuticalcomposition to a patient diagnosed with vascular thrombosis (forexample, venous thromboembolism), inflammatory arthritis, systemic lupuserythematosus (SLE), atherosclerosis, infection (for example, viralinfection), sepsis, acute lung injury arthritis (ALI) and acuterespiratory distress syndrome (ARDS).

In some embodiments, the present invention provides a method ofinhibiting signs of inflammation, comprising exposing to a samplecomprising inflammatory cells a composition comprising a Poly-Aparticle, wherein said exposing results in inhibition of signs ofinflammation. In certain embodiments, the sample is from a human. Inparticular embodiments, the human is diagnosed with vascular thrombosis,inflammatory arthritis, systemic lupus erythematosus (SLE),atherosclerosis, sepsis, acute lung injury arthritis (ALI) and acuterespiratory distress syndrome (ARDS). In other embodiments, the sampleis a sample selected from the group consisting of a blood sample, aserum sample, a plasma sample, a saliva sample, a urine sample, asynovial fluid sample, a cartilage sample, and a tissue sample.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising at least one Poly-A particle. In certainembodiments, the Poly-A particle is a targeted Poly-A particle. In otherembodiments, the targeted Poly-A particle is a Poly-A VTP. In furtherembodiments, the Poly-A particle is a nTP. In particular embodiments,the Poly-A particle is biocompatible and biodegradable. In a givenembodiment, that Poly-A particle comprises a bioactive molecule.

In some embodiments, the present invention provides a pharmaceuticalcomposition consisting of at least one Poly-A particle and at least onepharmaceutically acceptable carrier. In some embodiments, the presentinvention provides a pharmaceutical composition wherein at least onePoly-A particle is made by the method of one or more or all of the stepsof: a) dissolving polyvinyl alcohol (PVA) with an average molecularweight of 20-70 kDA in water to generate a 1 wt % PVA solution of pH6-7; b) dissolving Poly-A in dichloromethane (DCM); c) adding thesolution comprising the Poly-A in the DCM to the PVA solution over atleast one hour during mixing at >4000 rpm to generate an emulsion; d)centrifuging the emulsion; e) aspirating a centrifuged solution from acentrifuged pellet; 0 resuspending the pellet in deionized water togenerate suspended Poly-A particles; g) washing the suspended Poly-Aparticles; h) lyophilizing the washed Poly-A particles; and i) freezingthe lyophilized Poly-A particles. In other embodiments, the Poly-Aparticle is modified to be a carrier of one or more hydrophobicbioactive compounds or drugs by adding the one or more hydrophobicbioactive compounds or drugs to the Poly-A polymer dissolved in saidDCM. In further embodiments, the Poly-A particle is modified to be acarrier of one or more hydrophilic bioactive compounds or drugs byadding the one or more of the hydrophilic bioactive compounds or drugsto a water phase that is emulsified into the Poly-A polymer dissolved insaid DCM, and emulsifying the drug-polymer emulsion in a solution of 1wt % PVA in water.

In some embodiments, the present invention provides a pharmaceuticalcomposition consisting of at least one PLGA particle and at least onepharmaceutically acceptable carrier. In some embodiments, the presentinvention provides a pharmaceutical composition wherein at least onePLGA particle is made by the method of one or more or all of the stepsof: a) dissolving polyvinyl alcohol (PVA) with an average molecularweight of 20-70 kDA in water to generate a 0.5 wt % PVA solution of pH5-6; b) dissolving PLGA (50:50 PLGA (molecular weight of, for example,6.4 kDA in dichloromethane (DCM); c) adding the solution comprising thePLGA in the DCM to the PVA solution over at least one hour during mixingat >4000 rpm to generate an emulsion; d) centrifuging the emulsion toremove larger particles; e) centrifuging the supernatant to collect ˜1.5um particles; 0 aspirating a centrifuged solution from a centrifugedpellet; g) resuspending the pellet in deionized water to generatesuspended PLGA particles in a solution; h) flash freezing said solution;i) lyophilizing said PLGA particles; and j) freezing said lyophilizedPLGA particles.

In some embodiments, the present invention provides a pharmaceuticalcomposition wherein at least one Poly-A VTP particle is made by themethod of one or more or all of the steps of: a) suspending Poly-Aparticles in 50 mM MES buffer; b) suspending the particles inNeutravidin solution in 50 mM MES; c) adding1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC)solution to the suspended particles; d) adding glycerine to the solutioncomprising the Poly-A particles; e) centrifuging the solution; f)resuspending the Poly-A particles in PBS; and g) incubating the solutioncomprising the Poly-A particles with biotinylated anti-ICAM-1 in PBS −/−with 2% BSA.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows a scanning electron microscopy (SEM) image of Poly-Aparticles fabricated via emulsion solvent evaporation (ESE) method after(A) 0 and (B) 10 days of hydrolytic degradation in water. (C) showsrelease of salicylic (SA) from degrading Poly-A spheres measured byfluorescence intensity of SA in the media over time.

FIG. 2: Shows P-selectin expression by resting human platelets with orwithout Poly-A particle incubation relative to agonist (ADP) activatedplatelets. Poly-A non-targeted microparticles do not activate humanplatelets, thereby demonstrating hemocompatibility.

FIG. 3: Shows neutrophil counts and cytokine concentrations in lungbronchoalveolar lavage fluid (BALF) of untreated (UT) andlipopolysaccharide (LPS)-treated (ALI) mice. PA=Poly-A particles,PS=Polystyrene particles. Mouse treatments are provided on the X axis.

FIG. 4: Shows bacterial counts in the blood (top), and TNF-αconcentrations in the BALF of P. aeruginosa infected mice (C57BL/6J)with no microparticles (MPs), or with Poly-A MPs 6 hrs or 18 hrs afterinfection.

FIG. 5: Shows neutrophil counts in the BALF of P. aeruginosa (Pa)infected mice (C57BL/6J) without or with Poly-A microparticle (MP)treatment at 6 hrs and 18 hrs after bacterial infection. BALF sampleswere harvested 24 hrs after infection.

FIG. 6: Shows accumulation of anti-intracellular adhesion molecule(ICAM)-1 targeted (shaded) and non-targeted PS MPs in the lung 24 hrafter tail vein injection.

FIG. 7: Shows proposed timelines for mice particle injections and tissueharvest relative to the timing of P. aeruginosa infection.

FIG. 8: Shows structures of polymers 1-3 of use in formulatingsalicylate-based Poly-A microspheres and their hydrolytic degradation ofSA release.

FIG. 9: Shows TNF-α secretion by alveolar macrophages cultured for 2 hrsor 24 hrs and then exposed to Poly-A or PS MPs relative to untreated(UT) macrophages.

FIG. 10: Shows Poly(lactic-co-glycolic acid) (PLGA) and Poly-Amicrosphere particle fabrication using single emulsion and solventevaporation.

FIG. 11: Shows PLGA and Poly-A microspheres and Zeta-potentials (mV).The Zeta potential is a function of the surface charge of a particlerelative to the nature of the medium that surrounds it. The valuecorrelates to how particles will interact with WBCs. The Zeta potentialvalue for Poly-A is similar to PLGA, but Poly-A has an added beneficialeffect in ALI/ARDS.

FIG. 12: Shows A) Poly-A particle degradation over time (hours), and B)concentration of released salicylic acid (SA) by incubating particles inplasma (Conc.=0.6 mg/mL).

FIG. 13: Shows injection timeline for a BALB/c model of lunginflammation after lipopolysaccharide (LPS) tracheal instillation. LPSinflames the lungs and gives rise to neutrophil infiltration. Two hoursafter particle administration, mice are euthanized and lungs are lavagedto collect cells in the airspaces. The supernatant is analyzed for thepresence of inflammatory markers.

FIG. 14: Shows BALF percent of monocytes and neutrophils without LPSinjury, percent of monocytes and neutrophils after LPS injury and notreatment, percents of monocytes and neutrophils after LPS injury andtreatment with Poly-A particles, and total BALF cell counts inuntreated, LPS injury only, and LPS injury/Poly-A particle treatment.BALB/c mice were induced to have ALI via instillation of LPS in thelungs. Untreated mice received no LPS. Data shown in “UntreatedMonocytes vs. Neutrophils” are from mice that received no LPS and noparticle treatment. Data shown in “LPS Monocytes vs. Neutrophils” arefrom mice that received LPS and no particle treatment. Neutrophilspredominate in the BALF. Data shown in “LPS+P Monocytes vs. Neutrophils”are from mice that were administered LPS, and then Poly-A particletreatment at 1 hr after LPS. The number of neutrophils in the lungs isreduced significantly. The bar graph provides total cell counts.

FIG. 15: Shows comparative IL-6, TNF-α, KC and IL-10 concentrations inuninjured/untreated mice, in LPS injured/untreated mice, and in LPSinjured/Poly-A particle treated mice (LPS+P).

FIG. 16: Shows comparative MCP-1, albumin, MIP2 and IP10 concentrationsin uninjured/untreated mice, in LPS injured/untreated mice, and in LPSinjured/Poly-A particle treated mice.

FIG. 17: Shows cell counts in BALF samples from untreated BALB/c mice,LPS injured BALB/c mice, LPS injured/PS—COOH particle treated BALB/cmice, LPS injured/Poly-A particle treated BALB/c mice, LPSinjured/aspirin treated BALB/c mice, and LPS injured/vehicle controltreated BALB/c mice averaged over multiple experiments. Poly-A particlesprovide protection from lung injury. (PA=Poly-A.)

FIG. 18: Shows cell counts in BALF samples from untreated mice, LPSinjured mice, LPS injured/PS—COOH particle treated mice, LPSinjured/Poly-A particle treated mice, LPS injured/aspirin treated mice,and LPS injured/vehicle control treated mice in a single experiment.

FIG. 19: Shows albumin concentrations and IL-6 concentrations in BALFsupernatants from untreated mice, LPS injured mice, LPSinjures/polystyrene (PS—COOH) particle treated mice, LPS injured/Poly-Aparticle treated mice, LPS injured/aspirin treated mice, and LPSinjured/vehicle control treated mice.

FIG. 20: Shows IL-10 and KC concentrations in BALF supernatants fromuntreated mice, LPS injured mice, LPS injured/polystyrene (PS—COOH)particle treated mice, LPS injured/Poly-A particle treated mice, LPSinjured/aspirin treated mice, and LPS injured/vehicle control treatedmice.

FIG. 21: Shows MCP1 and MIP2 concentrations in BALF supernatants fromuntreated mice, LPS injured mice, LPS injured/polystyrene (PS—COOH)particle treated mice, LPS injured, Poly-A particle treated mice, LPSinjured/aspirin treated mice, and LPS injured/vehicle control treatedmice.

FIG. 22: Shows TNF-α concentrations in BALF supernatants from untreatedmice, LPS injured mice, LPS injured/polystyrene (PS—COOH) particletreated mice, LPS injured/Poly-A particle treated mice, LPSinjured/aspirin treated mice, and LPS injured/vehicle control treatedmice.

FIG. 23: Shows a representative experimental timeline for collection ofBALF samples after ALI caused by LPS instillation in C57BL/6J micewithout particle treatment.

FIG. 24: Shows comparative intervals for neutrophil infiltration in theBALF after LPS injury (ALI) vs. percent of immune cells and totalneutrophil counts in blood in sepsis for C57BL/6J.

FIG. 25: Shows a representative experimental timeline for collection ofBALF samples after ALI caused by LPS instillation using 30 mg/kginjections of Poly-A particles.

FIG. 26: Shows the comparative impacts of Poly-A particle administrationat 4 hrs after LPS on changes in the percent of immune cells and totalneutrophil counts in BALF samples relative to treatments with LPS only,LPS plus Polystyrene (PS) particles, LPS plus PLGA particles, and LPSplus Poly-A particles.

FIG. 27: Shows that Poly-A particles reduce albumin concentrations inBALF samples in LPS injured/PS particle treated mice, and in LPSinjured/Poly-A particle treated mice similar to uninjured, untreatedmice compared to LPS injured/untreated mice, and LPS injured/PLGAparticle treated mice when treatment is provided 4 hrs after LPS injury.PLGA particles increase lung leakiness, whereas Poly-A particles protectthe lungs from LTP injury as reflected in lower BALF albuminconcentrations.

FIG. 28: Shows that the total cell count in BALF samples are highest inLPS injured/untreated mice and in LPS injured/PLGA particle treatedC57BL/6J mice compared to LPS injured/Poly-A particle treated mice whentreatment is provided 2 hrs after LPS injury. PLGA particles have nobenefit on total BALF cell counts, whereas Poly-A particles protect thelungs from cellular infiltration when given 2 hrs after infection.

FIG. 29: Shows that there is no difference in treatment of LPS injuredmice between no treatment, treatment with PLGA particles, and treatmentwith Poly-A particles when treatment is provided 6 hrs after LPS injuryin C57BL/6J mice.

FIG. 30: Shows a representative experimental timeline for mice P.aeruginosa infection, Poly-A particle administration, and collection ofsamples for BALF assays and blood colony forming unit (CFU) counts.

FIG. 31: Shows IL-6 and IL-10 comparative concentrations in miceinfected with P. aeruginosa without Poly-A particle administration, withPoly-A particle administration at 6 hrs after infection, and at 18 hrsafter infection, and in no infection control mice. Markers ofinflammation are reduced with treatment with Poly-A particles, withgreater benefit when particles are administered at 18 hours afterinfection.

FIG. 32: Shows KC and MIP2 comparative concentrations in mice infectedwith P. aeruginosa without Poly-A particle administration, with Poly-Aparticle administration at 6 hrs after infection, and at 18 hrs afterinfection, and in no infection control mice. Markers of inflammation arereduced with treatment with Poly-A particles, with greater benefit whenparticles are administered at 18 hrs after infection.

FIG. 33: Shows MCP-1 and TNF-α comparative concentrations in miceinfected with P. aeruginosa without Poly-A particle administration, withPoly-A particle administration at 6 hrs after infection, and 18 hrsafter infection, and in no infection control mice. Markers ofinflammation are reduced with treatment with Poly-A particles, withgreater benefit when particles are administered at 18 hours afterinfection.

FIG. 34: Shows comparative total BALF cell counts and total BALFneutrophil counts in mice infected P. aeruginosa without Poly-A particleadministration, with Poly-A particle administration at 6 hrs afterinfection, and 18 hrs after infection, and saline injection after 18 hrsafter infection in no infection control mice. Treatment with Poly-Aparticles reduces the number of neutrophils with a greater reductionobserved with administration at 18 hrs vs. 6 hrs after infection.

FIG. 35: Shows comparative total BALF cell counts, P. aeruginosa 19660log BAL CFU/mL counts, and P. aeruginosa 19660 log blood CFU/mL countsat 12 hrs, 18 hrs, 24 hrs, 30 hrs and 36 hrs after P. aeruginosainfection and no treatment. White cells and bacteria increase in thelungs after infection, as do bacterial CFU counts in blood.

FIG. 36: Shows comparative P. aeruginosa 19660 log BAL CFU/mL counts,and P. aeruginosa 19660 log blood CFU/mL counts at 24 hrs afterinfection in untreated mice, in mice treated with intravenous Poly-Aparticles intravenously, and in mice treated with intravenous salineassayed at 18 hrs. Poly-A particles decrease P. aeruginosa 19660 log BALCFU/mL counts, and P. aeruginosa 19660 log blood CFU/mL counts at 24 hrsafter infection.

FIG. 37: Shows the comparative benefit of Poly-A particle treatment ondaily survival to 10 days after P. aeruginosa infection. Mice wereinfected with P. aeruginosa and at 18 hrs after infection. A firstsubset was treated with Poly-A particles (“Particle A i.v.”), and asecond subset was untreated. Mice in the Poly-A treated subset had 60%survival compared to 40% survival in the untreated group.

FIG. 38: Shows flow cytometry histogram plots of Poly-A-Avidin andPoly-A-anti-ICAM-1 VTP.

FIG. 39: Shows the impact of Poly-A particle injection on P. aeruginosalung infection in C57BL/6J mice. Total cells in BALF at 24 hrspost-infection after 6 hr or 18 hr Poly-A injection, 18 hr PLGAinjection, or 18 hr polystyrene injection are compared between infectedmice and saline control.

FIG. 40: Shows that the concentration of IgM in BALF is reduced infectedmice by treatment with Poly-A particles.

FIG. 41: Shows comparative concentrations of inflammatory cytokines byELISA quantification of (a) TNF, (b) 11-6, (c) IL-10, and (d) IP-10produced by alveolar macrophages in response to LPS treatment for 3 or24 hours, with or without the addition of Poly-A or polystyrene (PS)particles.

FIG. 42: Shows post-infection survival for P. aeruginosa infected mice(N=5 for each group) with and without 18-hour Poly-A MP injection. 2E8particles were injected in each mouse for every particle type.

DEFINITIONS AND METHODS

While the invention will be described in conjunction with certainrepresentative embodiments, it will be understood that the invention isnot limited to these illustrative examples. One skilled in the art willrecognize many methods and materials similar or equivalent to thosedescribed herein may be used in the practice of the present invention.The present invention is in no way limited to the methods and materialsdescribed.

Unless defined otherwise, technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which this invention belongs. Definitions of common terms inmolecular biology may be found in Benjamin Lewin, Genes V, published byOxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.(eds.), The Encyclopedia of Molecular Biology, published by BlackwellScience Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.),Molecular Biology and Biotechnology: a Comprehensive Desk Reference,published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Althoughany methods, devices, and materials similar or equivalent to thosedescribed herein can be used in the practice of the invention, certainmethods, devices, and materials are described herein. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art(s) to which this invention belongs. Although any methods,devices, and materials similar or equivalent to those described hereincan be used in the practice or testing of the invention, the preferredmethods, devices and materials are now described.

As used in this disclosure, including the appended claims, the singularforms “a,” “an,” and “the” include plural references, unless the contentclearly dictates otherwise, and are used interchangeably with “at leastone” and “one or more.” Thus, reference to “a Poly-A MP” includesmixtures of Poly-A MPs, and the like.

As used herein, the term “about” represents an insignificantmodification or variation of the numerical value such that the basicfunction of the item to which the numerical value relates is unchanged.

As used herein, “protein” is used synonymously with “peptide,”“polypeptide,” or “peptide fragment.” A “purified” polypeptide, protein,peptide, or peptide fragment is substantially free of cellular materialor other contaminating proteins from the cell, tissue, or cell-freesource from which the amino acid sequence is obtained, or substantiallyfree from chemical precursors or other chemicals when chemicallysynthesized.

As used herein, “inflammatory disease” refers to a disease or conditioninvolving an inflammatory response. The inflammatory response may beacute and/or chronic. In some embodiments, chronic inflammation involvesan increase neutrophil number and/or activity. Non-limiting exemplaryinflammatory diseases that may be treated with Poly-A particlesdescribed herein include rheumatoid arthritis, juvenile idiopathicarthritis, systemic-onset juvenile idiopathic arthritis, osteoarthritis,sepsis, asthma, interstitial lung disease, inflammatory bowel disease,systemic sclerosis, intraocular inflammation, Grave's disease,endometriosis, systemic sclerosis, adult-onset Still disease, amyloid Aamyloidosis, polymyalgia rheumatic, remitting seronegative symmetricalsynovitis with pitting edema, Behcet's disease, uveitis,graft-versus-host diseases, venous thromboembolism, ALI/ARDS andTNFR-associated periodic syndrome.

As used herein, “infection” refers to a disease or condition caused by apathogen, such as a bacteria, virus, fungus, etc. Non-limiting exemplaryinfections that may be treated with the Poly-A particles describedherein include bacterial, viral, fungal, rickettsia′, and parasiticinfections. In some embodiments, the viral infection is a respiratoryvirus infection including, for example, influenza virus infection,corona virus infection (e.g. severe acute respiratory syndromecoronavirus 2 (SARS-CoV2)), and respiratory syncytial viral infection.

As used herein, “autoimmune disease” refers to a disease or conditionarising from an inappropriate immune response against the body's owncomponents, such as tissues and other components. In some embodiments,neutrophil numbers and activity are elevated in autoimmune disease.Non-limiting exemplary autoimmune diseases that may be treated with thePoly-A particles described herein include systemic lupus erythromatosus,systemic sclerosis, polymyositis, vasculitis syndrome including giantcell arteritis, takayasu aeteritis, cryoglobulinemia,myeloperoxidase-antineutrophil cytoplasmic antibody-associatedcrescentic glomerulonephritis, rheumatoid vasculitis, Crohn's disease,relapsing polychondritis, acquired hemophilia A, and autoimmunehemolytic anemia.

As used herein, a “neutrophil mediated condition or disease” refers to adisease or condition in which at least some of the symptoms and/orprogression of the disease or condition is caused neutrophilaccumulation and/or activity. Non-limiting exemplary neutrophil mediateddiseases or conditions include inflammatory diseases, malignant diseases(including cancer and cancer-related conditions), infections, andautoimmune diseases. Further non-limiting exemplary neutrophil mediateddiseases include, but are not limited to, Castleman's disease,ankylosing spondylitis, coronary heart disease, cardiovascular diseasein rheumatoid arthritis, pulmonary arterial hypertension, chronicobstructive pulmonary disease (COPD), atopic dermatitis, psoriasis,sciatica, venous thrombosis, type II diabetes, obesity, giant cellarteritis, acute graft-versus-host disease (GVHD), non-ST elevationmyocardial infarction, anti-neutrophil cytoplasmic antibody (ANCA)associated vasculitis, neuromyelitis optica, chronic glomerulonephritis,and Takayasu arteritis.

As used herein, “modulate” means to alter, either by increasing ordecreasing, the number and/or activity of a cell. The term “inhibit”, asused herein, means to prevent or reduce cell number and/or activity. Aused herein the cell that is modulated is a neutrophil.

As used herein, the term “bioactivity” indicates an effect on one ormore cellular or extracellular process (e.g., via binding, signaling,etc.) which can impact physiological or pathophysiological processes.

As utilized herein, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of a federal or a state government orlisted in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals and, more particularly, in humans. Theterm “carrier” and/or “formulation” refers to a diluent, adjuvant,excipient, or vehicle with which the therapeutic is administered andincludes, but is not limited to, such sterile liquids as water and oils.

A “pharmaceutically acceptable salt” or “salt” is a product of adisclosed compound that contains an ionic bond and is typically producedby reacting the disclosed compound with either an acid or a base,suitable for administering to an individual. A pharmaceuticallyacceptable salt can include, but is not limited to, acid addition saltsincluding hydrochlorides, hydrobromides, phosphates, sulphates, hydrogensulphates, alkylsulphonates, arylsulphonates, arylalkylsulfonates,acetates, benzoates, citrates, maleates, fumarates, succinates,lactates, and tartrates; alkali metal cations such as Li, Na, K, alkaliearth metal salts such as Mg or Ca, or organic amine salts.

A “pharmaceutical composition” is a formulation comprising a Poly-Apolymer particle in a form suitable for administration to an individual.A pharmaceutical composition is typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include, but are not limited to, oral and parenteral,e.g., intravenous, intradermal, subcutaneous, inhalation, topical,transdermal, transmucosal, intra-articular, intra-ocular, and rectaladministration.

As used herein, the term “therapeutically effective amount” generallymeans the amount necessary to ameliorate at least one symptom of adisorder or condition to be prevented, reduced, or treated as describedherein. The phrase “therapeutically effective amount” as it relates tothe Poly-A particle of the present disclosure means the Poly-A particledosage that provides the specific pharmacological response for which thePoly-A particle is administered in a significant number of individualsin need of such treatment. It is emphasized that a therapeuticallyeffective amount of a Poly-A particle that is administered to aparticular individual in a particular instance will not always beeffective in treating the conditions/diseases described herein, eventhough such dosage is deemed to be a therapeutically effective amount bythose of skill in the art.

As used herein, the term “second agent” refers to a therapeutic agentother than a Poly-A particle in accordance with the present invention.In certain instances, the second agent is an anti-inflammatory agent.

As used herein, the term “sepsis” refers to the presence of the presenceof harmful microorganisms in the blood.

The term “co-administration” refers to the administration of at leasttwo agent(s) (e.g., a Poly-A particle) or therapies to a subject. Insome embodiments, the co-administration of two or more agents/therapiesis concurrent. In other embodiments, a first agent/therapy isadministered prior to a second agent/therapy. Those of skill in the artunderstand that the formulations and/or routes of administration of thevarious agents/therapies used may vary. The appropriate dosage forco-administration can be readily determined by one skilled in the art.In some embodiments, when agents/therapies are co-administered, therespective agents/therapies are administered at lower dosages thanappropriate for their administration alone. Thus, co-administration isespecially desirable in embodiments where the co-administration of theagents/therapies lowers the requisite dosage of a known potentiallyharmful (e.g., toxic) agent(s).

The term “combination therapy” includes the administration of ananti-inflammatory agent (e.g., a Poly-A particle) and at least a secondagent as part of a specific treatment regimen intended to provide thebeneficial effect from the co-action of these therapeutic agents. Thebeneficial effect of the combination includes, but is not limited to,pharmacokinetic or pharmacodynamic co-action resulting from thecombination of therapeutic agents. Administration of these therapeuticagents in combination typically is carried out over a defined timeperiod (usually minutes, hours, days or weeks depending upon thecombination selected). “Combination therapy” may, but generally is not,intended to encompass the administration of two or more of thesetherapeutic agents as part of separate monotherapy regimens thatincidentally and arbitrarily result in the combinations of the presentinvention. “Combination therapy” is intended to embrace administrationof these therapeutic agents in a sequential manner, that is, whereineach therapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single capsule or injectionhaving a fixed ratio of each therapeutic agent or in multiple, singlecapsules or injections for each of the therapeutic agents. Sequential orsubstantially simultaneous administration of each therapeutic agent canbe affected by any appropriate route including, but not limited to, oralroutes, intravenous routes, intramuscular routes, intra-articularroutes, corneal routes, topical routes, and direct absorption throughmucous membrane tissues. The therapeutic agents can be administered bythe same route or by different routes. For example, a first therapeuticagent of the combination selected may be administered by intravenousinjection while the other therapeutic agents of the combination may beadministered orally. Alternatively, for example, all therapeutic agentsmay be administered orally, or all therapeutic agents may beadministered by intravenous injection. The sequence in which thetherapeutic agents are administered is not narrowly critical.“Combination therapy” also can embrace the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients and non-drug therapies (e.g., surgery orradiation treatment). Where the combination therapy further comprises anon-drug treatment, the non-drug treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and non-drug treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the non-drug treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (as well as fractions thereofunless the context clearly dictates otherwise). Any concentration range,percentage range, ratio range, or integer range is to be understood toinclude the value of any integer within the recited range and, whenappropriate, fractions thereof (such as one tenth and one hundredth ofan integer), unless otherwise indicated. Also, any number range recitedherein relating to any physical feature, such as polymer subunits, sizeor thickness, are to be understood to include any integer within therecited range, unless otherwise indicated.

DETAILED DESCRIPTION

Provided herein are methods of treatment, compositions, systems and kitsusing polymer particles as restraints of neutrophil function. Suchmethods include, but are not limited to, methods of preventing,treating, and/or ameliorating inflammatory diseases, infections,autoimmune diseases, malignant diseases, and other diseases orconditions in which neutrophils may be implicated. In some embodiments,polymer particles are useful for diagnosing neutrophil related diseasesor conditions.

ALI/ARDS

Numerous and complex pathologies lead to ALI/ARDS. Either a direct or anindirect injury initiates ALI/ARDS with no differences in the overallmortality. A direct pulmonary injury occurs with pathologies that startin the lungs, e.g., pneumonia, that induce activation of lungmacrophages and are followed by damage to the lung epithelia. Thecascade of inflammatory events in the lungs then triggers inflammationof the lung endothelium, and the recruitment of primary leukocytes fromthe blood into the lungs, thereby propagating the injury. An indirectinjury arises from a pathology outside the lungs e.g., sepsis or trauma,in which the disease results in systemic inflammation that initiatesrapid leukocyte migration into the lungs. Migration of leukocytesdamages the lung endothelium and eventually the lung epithelia.Regardless of the primary cause of ALI, the outcome is a damagedalveolar epithelium, leading to the rapid infiltration of the alveoli byimmune cells and protein-rich fluid causing compromised lung function.

Neutrophils in ALI/ARDS

The inflammatory cascade in ALI/ARDS triggers the capillary endotheliumto express leukocyte adhesion molecules (LAMs) that facilitate the rapidmigration of circulating blood neutrophils. LAMs including selectin,intracellular adhesion molecule (ICAM)-1 and vascular cell adhesionmolecule (VCAM)-1 promote rapid migration of circulating white bloodcells (WBCs) and leukocytes in the lung tissue. Neutrophils are the mostabundant WBCs comprising 60%-70% of WBC counts, and are the mostefficient first responders in acute inflammation. Accordingly,neutrophils are the primary cell type in bronchoalveolar lavage fluid(BALF) from ARDS patients, and disease severity correlates with theconcentration of neutrophils in BALF samples. Neutrophils are theprimary perpetrator of inflammation in ALI/ARDS causing damage in atleast 2 ways. First, excess migration of neutrophils into the lungscontributes to destruction of the alveolar-capillary barrier that leadsto edema in lungs. Second, neutrophils accumulate in the lung tissue andalveolar airspace, and release damaging pro-inflammatory andpro-apoptotic factors that impact resident cells and that give rise tofurther damage to the lungs. Halting the negative contribution ofneutrophils provides an opportunity for targeted treatment of ALI/ARDSand other inflammatory conditions. Drugs that block or suppressexpression of LAMs e.g., lisophylline and talactoferrin, have failed inclinical trials. CD11b/CD18 (Mac-1) integrin is important in Escherichia(E.) coli LPS and Pseudomonas (P.) aeruginosa-induced ALI, but blockingCD18 reduces neutrophil lung migration by only 60%.

In experiments conducted in the course of development of embodiments ofthe present invention, non-targeted and vascular-targeted nanoparticlesand microparticles (VTPs) (e.g., particles with surfaces bound with anantibody or ligand that targets proteins expressed on the vascular wallincluding, for example, anti-E-selectin antibody, anti-ICAM-1 antibody,anti-VCAM-1 antibody, and the like, and other peptides and carbohydratesthat bind selectins and LAMs) have been discovered to passively (i.e.,without an active pharmaceutical ingredient (API)), and rapidly blockneutrophil accumulation into inflamed tissue in ALI/ARDS, therebyhalting their destructive role in ALI/ARDS. Polystyrene (PS) VTPs inhuman blood flow interact with and reduce vascular wall adhesion ofneutrophils to a monolayer of activated endothelial cells (ECs) in vitroin a parallel plate flow chamber. Selectin-targeted, polystyrene (PS)VTPs provide nearly 100% reduction in neutrophil adhesion by physicalcoverage of the EC surface, thus blocking neutrophil attachment. At highparticle concentrations, neutrophil adhesion is prevented both byphysical coverage of the EC surface, and by free stream particle-cellinteractions (˜55% reduction in WBC adhesion with non-targeted,non-adhesive particles at 108 particles/ml of blood), demonstrating thatPS-VTPs in human blood alter neutrophil-vascular wall adhesion, andproviding a new opportunity for anti-inflammatory therapy in ALI whereinrapid intervention is desirable. In a lipopolysaccharide (LPS) mousemodel of ALI, LPS administered to the lungs of healthy mice inducesrapid recruitment of neutrophils into the lungs. When LPS-ALI mice aretreated with 2 μm PS microparticles administered 1 hour after LPSinstillation via tail vein injection at 30 mg/kg, the total lung lavageneutrophil count drops by 93% to 2.9×10⁶ from the LPS-only mice.LPS-treated mice that received 500 nm PS-VTPs had a drop in total BALFneutrophil to 6.4×105, equaling a 98% decrease from the LPS-only mice.Both PS particle-treated groups were not statistically different fromthe untreated (no LPS) mice. Although both particle sizes induced thesame level of neutrophil reduction in LPS-treated mice, microparticlesmore efficiently reduced lung neutrophil count compared to nanoparticlesin view of 64 times more nanoparticles injected by number equivalent todosing by mass. LPS instillation alone does not result in migration ofmonocytes or change in the absolute number of macrophages in the lungswith or without particle injection. Although the methods, compositions,kits and systems of the present invention are not restricted to aparticular mechanism, polymer particles appear to achieve therapeuticbenefit against unwanted neutrophil accumulation in the airspace inALI/ARDS through physical interactions that reduce leukocyte adhesion toinflamed endothelium (e.g., collisions in blood flow that disruptleukocyte adhesion, specific binding to LAMs expressed by theendothelium in inflamed tissue in competition with leukocytes forbinding sites, particle phagocytosis/internalization that altersleukocyte phenotypes, and diversion of neutrophils from the lung andblood to the liver). These features stand apart from the use of polymerparticles as a drug or bioactive compound carrier, i.e., VTCs,configured for delivery of a bioactive molecule (e.g. a protein orpeptide antigen directed towards the cells of adaptive immunity), or apharmacologic molecule. Thus, polymer particles that target neutrophilsprovide previously unknown therapies for neutrophil-mediatedinflammatory and other conditions, including vascular thrombosis,inflammatory arthritides, systemic lupus erythematosus (SLE),atherosclerosis, sepsis and ALI/ARDS.

The present invention provides polymer particles formed from, forexample, a biodegradable, biocompatible Poly-A polymer that blockneutrophil migration. Non-toxic degradation product of the Poly-Apolymer (i.e., salicylic acid) is itself anti-inflammatory, with theadded benefit of Poly-A particles for treatment in ALI/ARDS e.g., Poly-Aparticles are targeted to block neutrophil migration into the lungairway in ALI/ARDS, and locally release salicylic acid to further treatlung injury. In this fashion, it is contemplated that local neutrophiladhesion at an inflammation site is halted, migration of neutrophilsinto lung tissue and the airspace is rapidly and efficiently preventedwith minimal system impact, host protective responses are preserved, andbiodegradation is rapid. Direct action of Poly-A particles onneutrophils in the blood vessels of the lungs and other tissues afterintravascular injection, rather than indirect blocking adhesion orsignaling molecules, ensures that Poly-A polymer particles functionirrespective of the primary direct or indirect cause of ALI/ARDS, orother conditions in which neutrophils participate in the pathogenesis,such as vascular thrombosis, inflammatory arthritides, systemic lupuserythematosus (SLE), atherosclerosis, infection and sepsis.

Poly-A Microparticles

FIG. 1c and FIG. 8 show structure of a Poly-A polymer wherein “R”corresponds to linkers that range from small molecular weight linearhydrocarbons to branched aliphatic hydrocarbons. The Poly-A polymerparticle is biocompatible (Reynolds, M. A., A. Prudencio, M. E.Aichelmann-Reidy, K. Woodward, and K. E. Uhrich. Non-steroidalanti-inflammatory drug (NSAID)-derived poly(anhydride-esters) in boneand periodontal regeneration. Curr Drug Deliv, 2007. 4(3): p. 233-9,Bryers, J. D., R. A. Jarvis, J. Lebo, A. Prudencio, T. R. Kyriakides,and K. Uhrich. Biodegradation of poly(anhydride-esters) intonon-steroidal anti-inflammatory drugs and their effect on Pseudomonasaeruginosa biofilms in vitro and on the foreign-body response in vivo.Biomaterials, 2006. 27(29): p. 5039-48.) and stable under dry storageconditions Deronde, B. M., A. L. Carbone, and K. E. Uhrich, StorageStability Study of Salicylate-based Poly(anhydride-esters). Polym DegradStab, 2010. 95(9): p. 1778-1782.) When placed in aqueous solutions, thepolymer degrades to release salicylic acid (SA), which retains itsanti-inflammatory properties. In some embodiments, emulsion solventevaporation (ESE) techniques are used to fabricate degradablemicrospheres (FIG. 1A) from the Poly-A polymer having adipic acid as thelinker, i.e., R═(CH₂)₄ and MW˜20 kDA. (FIG. 8.) 20 mg of the Poly-Apolymer (Mw=˜20 kDa) is dissolved in 20 mL dichloromethane (oil phase)and the solution is emulsified into a solution of 1 wt % PVA in water(75 ml; aqueous phase). The oil phase is slowly injected via a syringeneedle, and the emulsion is stirred continuously for up to 2 hrs,allowing for hardening of the oil droplets. The resultant Poly-Aparticles are washed twice via centrifugation and dried vialyophilization. Particles are stored at −40° C. until use. The generatedPoly-A particles undergo hydrolytic degradation (FIG. 1C), and sustainedrelease of salicylic acid (SA) (FIG. 1C).

In some embodiments, Poly-A particles of the present invention arespherical. In certain embodiments, the Poly-A particles range from 100nm to 2 um in diameter. In particular embodiments, Poly-A spheres arefabricated with the polymer having the adipic acid linker (R═(CH₂)₄) anda molecular weight (Mw) of ˜20 kDa via the oil-in-water ESE method asdescribed previously. In other embodiments, the Poly-A particles arenon-spherical, and/or irregular in shape and surface morphologyincluding, for example, rods, ovals, stars, cones, cubes and the like.In further embodiments, the Poly-A particles in a single administrationare uniform in size, shape and surface morphology. In still furtherembodiments, the Poly-A particles in a single administration arenon-uniform in size, shape and surface morphology. In some embodiments,fabrication parameters, e.g., emulsification speed and oil phase polymerconcentration, are adjusted to achieve the desired average Poly-Aparticle sizes e.g., 200 nm and 2 μm. Scanning electron microscopy (SEM)images of the dried particles are used to evaluate particle surfacemorphology, and the particle size and zeta potential (ZP; a measure ofsurface charge) determined using a Malvern Zetasizer Nano-ZS. Inparticular embodiments, Poly-A particle degradation profiles aredetermined in phosphate buffer (PBS) and plasma at pH 7.4 and 37° C. viaa spectrophotometer (FIG. 1C), and changes in the solution pH aremonitored as the Poly-A particles degrade. In additional embodiments,Poly-A particle dimensions, morphology, uniformity and shape arequantified by flow cytometry, and optimized for the capacity to bindhuman umbilical vein endothelial cells from the flow of whole blood(e.g., human whole blood) to predict in vivo functionality.

In some embodiments, the hemo-compatibility of Poly-A particles withhuman cells is optimized in vitro. In certain embodiments, the potentialfor Poly-A particle toxicity in blood is evaluated and optimized viaassays of platelet activation and hemolysis. For platelet activationassays, Poly-A particles are incubated for 1 hr in platelet rich plasma(PRP) obtained via centrifugation of whole blood. The PRP samplesexposed to Poly-A particles are then stained with anti-CD41/61 (PE) andanti-CD62P (APC) to determine P-selectin expression via flow cytometry.P-selectin expression on resting platelets is minimal, and its highexpression on platelets is a sign of activation. For hemolysis assays,Poly-A particles are with isolated human RBCs in PBS buffer for 15, 30,60 and 120 min, after which the sample is centrifuged to pellet intactRBCs and particles. The supernatant from the Poly-A particle incubatedsample is evaluated for hemoglobin concentration as an indication of RBClysis via spectrophotometer. Non-targeted Poly-A microparticles do notactivate human platelets (FIG. 2), or induce hemolysis when placed inhuman blood.

In some embodiments, elements of the Poly-A particles of the presentinvention are optimized for preferred in vivo biodistribution andbiocompatibility by visualization of Poly-A microparticle adhesion toinflamed venules in experimental animal models of human diseases andconditions, together with healthy control animals. Adherence to inflamedvessel wall in vivo is detected with a fluorescent label within thePoly-A particle matrix. Using intravital microscopy (IVM) to visualizethe adhesion of Poly-A particles, their impact on the adhesion ofcirculating neutrophils is evaluated in vivo. Healthy C57BL/6J mice ofmixed sex (50:50) are used. In control assays, (1) Poly-A particles areobserved in non-inflamed mice (negative), and neutrophil adhesion isquantified in mice with mesentery inflammation but no particles (i.e.,vehicle only) injected. Poly-A particles in mice with inflammation areevaluated. Poly-A particles are targeted via anti-ICAM-1 (YN1/1.7.4;murine) antibody at 10,000 sites/μm². This antibody density issufficient to mediate firm arrest of microspheres to the inflamed vesselwall in vivo. Particles are injected in sterile PBS at ˜15-mg/kg, toyield a human equivalent dose of ˜1.2-mg/kg or ˜45-mg/m² which issufficient for reducing neutrophils in BALF in ALI mice. This dosealigns with the preclinical dosages evaluated in mice, and Phase IIhuman clinical trials have routinely employed between 35 and 50 mg/m²dose of pegylated liposomal doxorubicin.

In a mesentery inflammation model, mice are anesthetized, and a lateraltail vein catheter is placed for intravenous injection of antibodies,particles and additional anesthetic reagents as needed. The mice areplaced on a microscope with a heated stage at 37° C., and the mesenteryis exteriorized to a glass coverslip via a midline incision. Followingvessel selection, to confirm that neutrophils are the predominant cellsblocked from adhering to the inflamed vessels, neutrophils are tagged invivo by direct injection of fluorescent Ly6G antibody (5 μg of Gr-1 or1A8) before particle injection. Acute inflammation is induced by topicalapplication of TNF-α-10 μL of 200 μg/mL in PBS. Particles are injectedat 10 min after TNF-α activation. The mesentery vessels are imaged forparticle and cell adhesion up to 60 min via a 25× oil objective,inverted fluorescence microscope (Zeiss Axio Observer Z1 MarianasMicroscope). Images are recorded continuously in brightfield and greenfluorescence every 10 ms using Slidebook 6 software. Data on Poly-Aparticle adhesion and neutrophil blocking is collected at <5, 10, 15,and 60 min after particle injection. At 60 min, multiple vessels areimaged within the same animal for up to 2 min each to ensure anyobserved particle adhesion and neutrophil blocking is not an artifact ofthe single vessel chosen for imaging. At the end of the IVM imaging,mice are euthanized, and the Poly-A particle distribution in vitalorgans is evaluated, e.g., in lungs, liver, heart, kidney, and spleen,and compared between the inflamed and non-inflamed mice. Whole-organscans rating relative total fluorescence are obtained using an OdysseyCLx Infrared Imaging System (LI-COR), and then single cell suspensionsof all organs are obtained via a collagenase homogenization analyzed viacytometry. Homogenized liver samples are evaluated for changes inleukocyte population and cytokines.

In some embodiments, elements of the Poly-A particles of the presentinvention are optimized for preferred in vivo blood circulation andclearance. Kinetics assays in healthy mice are conducted wherein mouseblood is sampled at 5 min, 15 min and 0.5, 1, 2, 4, 6, 12, and 24 hrafter Poly-A particle injection (at, for example) 15 mg/kg), andevaluated for particle counts in order to characterize the clearancerate of Poly-A particles from the bloodstream. In certain embodiments, aPoly-A particle is made with an ICAM-1 antibody, and elements of thePoly-A particles are evaluated and compared. Control mice comprise micetreated with PBS only, and mice treated with non-targeted, 2 μm Poly-Aparticles. At the desired time, 10 μL of blood are collected and scannedfor Poly-A particles (e.g., by fluorescent scanning) on an Odyssey CLxInfrared imaging system. Values for pharmacokinetic parameters such asplasma half-life, distribution volume, and clearance are obtained fromthe plot of plasma concentration versus time profiles using thePKSolver, with the data fitted with a 2-compartment model. At 24 hrsafter particle injection, mice are euthanized, and particle distributionand cytokine levels are evaluated in critical organs that have beenextracted. Portions of the vital organs are subjected to hematoxylin andeosin (H&E) staining and are blindly scored by a clinical veterinarypathologist for any sign of tissue damage/inflammation associated withinjection of Poly-A particles.

In some embodiments, elements of the Poly-A particles of the presentinvention are optimized for preferred profiles of biocompatibility inhealthy mice. Poly-A particle injections described above are performed,and blood cell counts are measured. Mice are euthanized either at 2-,30-, 60-min after particle injection, or are returned to their cages andobserved for weight and behavioral changes over 7 days relative to theirbaseline before particle injection. The time points are chosen based onevidence that the neutrophil counts return to baseline by 1 hr afterparticle injection when particles are cleared from the bloodstream. Atthe targeted time interval, mice are euthanized via cardiac puncture forblood collection and vital organs removed. Blood for cell counts(platelets, neutrophils, monocytes, and lymphocytes), and hematocrit andhemoglobin levels relative to PBS control mice are measured, togetherwith levels of platelet activation and cellular apoptosis. The weight ofeach organ is recorded, and particle composition is assessed in tissuehomogenates. Histology is used to detect whether or not there is injuryor scaring in vital organs. Signs of inflammation are evaluated viameasurement of cytokine expression levels. Experiments conducted in thecourse of development of the present invention show that (1) Poly-Aparticles bind to the inflamed vessel and reduce neutrophil adhesion,and (2) Poly-A particles display minimal toxicity in mice; 100% of micewith LPS-induced ALI survive to at least 48 hrs after Poly-A particleinjection. In other embodiments, anti-ICAM-1 coated Poly-A is used. Inother embodiments, alternative routes or administration are usedincluding, for example, intravascular administration, intra-arterialadministration, intravenous administration, catheter-directedadministration, pulmonary artery catheter directed administration,interventional radiology administration, ultrasound guidedadministration, MRI guided administration, surgically guidedadministration, laparoscopically guided administration,bronchoscopically guided administration, intratracheal administration,intramuscular administration, subcutaneous administration, enteraladministration, rectal administration, inhaled administration,intraperitoneal administration, intra-articular administration,intraspinal administration, intracerebroventricular administration,intravessicular administration, intraparenchymal administration, and thelike. In other embodiments, samples are incubated with an antibodyagainst the isotype of the anti-ICAM-1 bound to Poly-A VTP.

In some embodiments, elements of the Poly-A particles of the presentinvention are optimized for their therapeutic benefit. In certainembodiments, the therapeutic impacts of Poly-A particles are compared ina Pseudomonas (P.) aeruginosa mouse model of ARDS. P. aeruginosa, agram-negative bacterium, is the second most common cause of pneumonia inhospitalized patients causing lung injury with a mortality rate of60-90% in mechanically ventilated patients. A mouse model of P.aeruginosa-induced lung injury replicates the histological andimmunological features of ARDS in humans; 46-51% of ARDS in humans arisefrom a pulmonary bacterial infection. The model evaluates the protectiveeffects of the Poly-A particles on lung injury along with possibledetrimental effects of the particles on innate protective responses.C57BL/6J mice of mixed sex (50:50) strain, and 19660 of P. aeruginosaobtained from ATCC, are used.

In some embodiments, elements of the Poly-A particles of the presentinvention are optimized for the timing of neutrophil transmigration andbarrier disruption. The time course of neutrophil emigration into thelungs is evaluated after P. aeruginosa administration in mice in theabsence of Poly-A particles to establish a baseline to guide the timingof Poly-A particle interventions. Experimental Example 2 shows theimportance of the “time after infection” at which Poly-A particles areinjected for achieving a significant therapeutic benefit (FIG. 5) linkedto the kinetics of neutrophil migration into the BALF and onset of lunginjury. Mice are infected with P. aeruginosa and the time at which miceare euthanized is varied e.g., at 6 hrs, 12 hrs, 18 hrs, 24 hrs, 30 hrsand 36 hrs after bacterial infection. The 36 hrs upper boundary ischosen in view of 20% survival at 48 hrs after P. aeruginosa infectionfor C57BL/6J mice, with ˜70% survival at 24 hr. The BALF, lung tissueand blood are analyzed for cellular content, bacterial (CFU) andinflammation markers. Leukocyte counts are obtained via flow cytometrywherein the BALF single-cell suspension or blood is stained with a panelof fluorescent antibodies to identify different cell populations.Neutrophils, monocytes, lymphocytes, macrophages and dendritic cells arestained in the BALF. Changes in the levels of inflammatory cytokines(e.g., IL-6, IL-12, MIP-2, MCP-1, IL-17 KC, IL-10, TNF-α, and IL-1β) inthe BALF supernatant and plasma via ELISA are compared. BALF albumin andIgM levels are quantified to evaluate alveolar epithelial integrity ininfected mice. Major organs, e.g., lungs, liver, heart, kidney, andspleen, are harvested and evaluated for bacterial CFUs, leukocytes andinflammatory cytokines. All observed cell counts and cytokine levels arecompared to the baseline in non-infected (vehicle only) mice.

In some embodiments, elements of the Poly-A particles of the presentinvention are optimized for the impact of timing of Poly-A particleadministration on reducing lung injury in P. aeruginosa-inducedALI/ARDS. The capacity of Poly-A particles to ameliorate lung injury inP. aeruginosa infected mice vs. the extent of injury characterized forthe “no particle” treatment is compared via evaluation of leukocytecount, cytokine levels, and bacteria CFU in the BALF and lungs, blood,and other major organs. VTPs of different diameter (e.g., 200 nm and 2μm VTPs with murine anti-ICAM-1 (YN1/1.7.4)) are evaluated. PS VTPscoated with this antibody are retained in lungs of ALI-mice for up to 24hrs unlike untargeted microparticles (FIG. 6). Mice are treated withVTPs (at, for example 15 mg/kg or other dose), injected at varying timesof 6 hrs, 12 hrs, 18 hrs, 24 hrs and 30 hrs after infection. Regardlessof the particle injection interval, mice are euthanized at a fixed 36hrs after interval after infection (FIG. 7). The capacity of Poly-Aparticles antibody-targeted (anti-ICAM-1 or anti-E-selectin oranti-VCAM-1) to enhance therapeutic impact is evaluated by comparisonwith administration of non-targeted, 2 μm Poly-A at the 18 hr pointshown to reduce BALF neutrophils and blood CFU in P. aeruginosa-infectedmice (Example 2). BALF cells are counted, and changes in albumin/IgMlevels, bacteria CFU, and inflammatory cytokines in the BALF supernatantrelative to the Poly-A injection time are measured. Lung tissue isassessed for particle content (i.e., by whole organ scan histology, andtissue homogenate), as well as for bacterial CFUs, cytokines andleukocyte count. The degree of lung injury, via blind scoring of H&Estained lung sections for epithelial thickening, airway epithelialnecrosis, and intra-alveolar edema is assessed for the different MPinjection times in comparison to the “no-microparticle” controls.

In some embodiments, elements of the Poly-A particles of the presentinvention are optimized for the time after particle injection at whichanimals are euthanized by comparison of the extent of lung injury andbacteria dissemination observed. Poly-A particles of 2 μm size areadministered at a fixed “time after infection” of 6 hrs and 18 hrs, andthe BALF and major organ cells/cytokine/CFU/particles counts are beanalyzed (i.e., mice are euthanized) at varying times of 6 hrs, 12 hrs,18 hrs, and 24 hrs after particle injection (FIG. 7). The liver isevaluated to determine if the injection of Poly-A particles with diverseelements promotes accumulation of neutrophils and particles and injuryto the liver in the ALI/ARDs mice. A whole organ scan of a fraction ofthe liver is used for detection of particle localization. A section ofthe liver is homogenized and evaluated for particles, leukocytepopulation, and expression of inflammation-associated cytokines. Thestructure of the liver tissue/cells is assessed via H&E staining, andliver function tests are performed (LFTs) on serum from the bloodcollected at euthanasia, including aspartate aminotransferase, alkalinephosphatase bilirubin, and alanine aminotransferase as a further measureof potential liver damage by particles in the ALI model. Controls arelivers from ALI/ARDs mice with no particle treatment and the vehiclecontrol.

In some embodiments, compositions of the Poly-A particles of the presentinvention are optimized for therapeutic benefit. The added benefits ofSA released from Poly-A particles are evaluated for changes in BALF cellcounts and cytokine levels by varying the rate of the Poly-A polymerdegradation, from non-degrading polymers to polymers that substantiallydegrade within days. FIG. 8 shows the structure of a Poly-A polymer,wherein “R” corresponds to linkers that range from linear hydrocarbons,e.g. (CH₂)₃, to branched aliphatics, and “n” is a polymer repeat unit.Particles fabricated from Poly-A polymers with the R1 and R2 adipic acidlinker degrade completely in 3 and 21 days, respectively(Rosario-Melendez, R., M. A. Ouimet, and K. E. Uhrich, Formulation ofsalicylate-based poly(anhydride-ester) microspheres for short- andlong-term salicylic acid delivery. Polym Bull (Berl), 2013. 70(1): p.343-351.) Particles with the R3 linker release 21% of their SA over 21days and reach complete degradation within 3.5 months. In certainembodiments, both “R” and “n” are tuned to achieve polymers forformulating 2 μm VTPS that substantially degrade in 3 days (R1), 7 (R2),21 (R2—baseline polymer), and >28 (R3) days. (Prudencio, A., R. C.Schmeltzer, and K. E. Uhrich, Effect of Linker Structure on SalicylicAcid-Derived Poly(anhydride-esters). Macromolecules, 2005. 38(16): p.6895-6901.) In some embodiments, a Poly-A particle degradation profileis determined via assays with microspheres that degrade in plasma.Particles are added to plasma at a concentration based on the fractionof particles injected identified in the lungs. Poly-A particles ofvarying degradation rates are fitted with anti-ICAM-1 antibody andinjected into P. aeruginosa infected mice at 18 hrs after infection.Mice are euthanized 24 hrs after infection. Blood, BALF, and tissues areharvested, and the impact of the Poly-A degradation rates on the cellcounts, cytokine and protein levels, and bacteria CFU are measured.Controls are infected mice treated with (1) aspirin in DMSO (100 ug/g),(2) DMSO only, (3) non-degrading polystyrene (PS) particles, and (4)degrading PLGA particles that release lactic acid instead of the SA.

In some embodiments, elements of the Poly-A particles of the presentinvention are optimized for neutrophil phagocytosis, e.g. rod shape.Surface protein expression and cytokine secretion by primary bloodleukocytes (e.g., neutrophils, monocytes, and lymphocytes) are measuredwith or without exposure to Poly-A particles. Mouse whole blood andisolated cells are incubated with, for example, 3- and >28-day degradingPoly-A particles for 1 hr. The particle-incubated cells are analyzed viaflow cytometry to measure the amount of Poly-A particles internalized,and the expression levels of LAMs, e.g., CD26L, CD15, CD11b, and CD66b,via fluorescent antibody staining. Additionally, leukocytes areevaluated for apoptosis markers, including annexin V, caspase, andphosphatidylserine. The supernatant is characterized via ELISA forpro-inflammatory factors released by isolated cells after phagocytosis.Controls are blood/cells exposed to PS particles and to vehicle (noparticles). The supernatant obtained from particle-treated, isolatedprimary cells are used to treat macrophages and dendritic cells (DCs)isolated from the lungs to identify factors secreted from Poly-Aparticle-exposed neutrophils (or monocyte or lymphocytes) associatedwith an anti-inflammatory phenotype compared to PS particle orvehicle-only controls. Mouse lung macrophage expression of inflammatorycytokines is not altered when incubated with Poly-A directly FIG. 9).Lung macrophages and DCs are isolated from dispersed lung digest cells.(Deng, J. C., G. Cheng, M. W. Newstead, X. Zeng, K. Kobayashi, R. A.Flavell, and T. J. Standiford, Sepsis-induced suppression of lung innateimmunity is mediated by IRAK-M. J Clin Invest, 2006. 116(9): p.2532-42.) Isolated macrophages and DCs are treated with LPS (lhr), orculture with P. aeruginosa (8 hr), before exposure to cell-releasedsupernatant for an additional 2 hr. Control assays are untreated and LPSonly/P. aeruginosa macrophages and DCs. The macrophage and DCsupernatant is evaluated factors secreted in ALI/ARDS, including IL-6,IL-10, IP-10, KC, MCP-1, MIP2, and TNF-α.

In some embodiments, polymer particles of the present invention comprisePoly-A polymer particles. In other embodiments, polymer particlescomprise PLGA polymer particles. Polymer particles of the presentinvention are not confined to Poly-A, PS or PLGA particles. Inparticular embodiments, polymer particles comprise additional polymersthat are biodegradable, targetable, and configured for microparticleand/or nanoparticle administration.

In some embodiments, the Poly-A particles of the present invention aregenerated using an emulsion solvent evaporation method. In certainembodiments, polyvinyl alcohol (PVA) (for example, 87-90% hydrolyzed PVAwith an average Mw of 20-70 dDa), is dissolved in water to provide a 1wt % PVA solution of pH 6-7. Poly-A (for example, 20 mg of Poly-A) isdissolved in dichloromethane (DCM), and the Poly-A in DCM oil phase isslowly added to the PVA water phase during mixing at 4000 rpm or greaterfor 1-2 hrs or longer. Particles generated are then centrifuged, and thePVA/water solution is aspirated. The pellet is resuspended in deionizedwater, washed, frozen under liquid nitrogen, lyophilized and stored at−20° C.

In some embodiments, the Poly-A particles of the present invention aremodified to be carriers of one or more bioactive compounds or drugs. Incertain embodiments, emulsion solvent evaporation (ESE) techniques areused to fabricate drug loaded degradable microparticles, for example,microspheres from the Poly-A polymer. For hydrophobic compounds anddrugs a single emulsion process is used wherein the compound or drug isadded to Poly-A polymer (Mw=˜20 kDa) dissolved in dichloromethane (oilphase), and the solution is emulsified into a solution of 1 wt % PVA inwater. The oil phase (poly-A and compound or drug) is slowly injectedvia a syringe needle, and the emulsion is stirred continuously for up to2 hrs, allowing the oil droplets to harden. The resultant Poly-Aparticles are washed twice via centrifugation and dried vialyophilization. Particles are stored at −40° C. until use. Forhydrophilic drugs, a double emulsion process (water in oil in water) isused. The compound or drug is added to a water phase that is emulsifiedinto the Poly-A polymer dissolved in dichloromethane. The drug-polymeremulsion is then be emulsified into a solution of 1 wt % PVA in water.

Pharmaceutical compositions that include at least one Poly-A particledescribed herein and at least one pharmaceutically acceptable carriermay also include one or more other active agents. The Poly-A particlesdescribed herein can be utilized in any pharmaceutically acceptabledosage form, including but not limited to injectable dosage forms,liquid dispersions, gels, aerosols, ointments, creams, lyophilizedformulations, dry powders, tablets, capsules, controlled releaseformulations, fast melt formulations, delayed release formulations,extended release formulations, pulsatile release formulations, mixedimmediate release and controlled release formulations, etc.Specifically, the Poly-A particles described herein can be formulated:(a) for administration selected from any of oral, pulmonary,intravenous, intra-arterial, intrathecal, intra-articular, rectal,ophthalmic, colonic, parenteral, intracisternal, intravaginal,intraperitoneal, local, buccal, nasal, and topical administration (forexample, dermatologic, mucosal, conjunctival and/or internal topicaladministration); (b) into a dosage form selected from any of liquiddispersions, gels, aerosols, ointments, creams, tablets, sachets andcapsules; (c) into a dosage form selected from any of lyophilizedformulations, dry powders, fast melt formulations, controlled releaseformulations, delayed release formulations, extended releaseformulations, pulsatile release formulations, and mixed immediaterelease and controlled release formulations; or (d) any combinationthereof.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can comprise one or more of the followingcomponents: (1) a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; (2) antibacterial agents such as benzylalcohol or methyl parabens; (3) antioxidants such as ascorbic acid orsodium bisulfate; (4) chelating agents such asethylenediaminetetraacetic acid; (5) buffers such as acetates, citratesor phosphates; and (5) agents for the adjustment of tonicity such assodium chloride or dextrose. The pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. A parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

Pharmaceutical compositions suitable for injectable use may includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition should be sterile and should be fluid to theextent that easy syringability exists. The pharmaceutical compositionshould be stable under the conditions of manufacture and storage andshould be preserved against the contaminating action of microorganismssuch as bacteria and fungi. The term “stable”, as used herein, meansremaining in a state or condition that is suitable for administration toa subject.

The carrier can be a solvent or dispersion medium, including, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol or sorbitol, and inorganic saltssuch as sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activereagent (e.g., a Poly-A particle) in an appropriate amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as desired, followed by filtered sterilization. Generally,dispersions are prepared by incorporating at least one Poly-A particleinto a sterile vehicle that contains a basic dispersion medium and anyother desired ingredient. In the case of sterile powders for thepreparation of sterile injectable solutions, exemplary methods ofpreparation include vacuum drying and freeze-drying, both of which willyield a powder of a Poly-A particles plus any additional desiredingredient from a previously sterile-filtered solution thereof. Oralcompositions generally include an inert diluent or an edible carrier.They can be enclosed, for example, in gelatin capsules or compressedinto tablets. For the purpose of oral therapeutic administration, thePoly-A particles can be incorporated with excipients and used in theform of tablets, troches, or capsules. Oral compositions can also beprepared using a fluid carrier for use as a mouthwash, wherein thecompound in the fluid carrier is applied orally and swished andexpectorated or swallowed. Pharmaceutically compatible binding agents,and/or adjuvant materials can be included as part of the composition.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from a pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, anebulized liquid, or a dry powder from a suitable device. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active reagents are formulated into ointments, salves, gels, orcreams as generally known in the art. The reagents can also be preparedin the form of suppositories (e.g., with conventional suppository basessuch as cocoa butter and other glycerides) or retention enemas forrectal delivery.

In some embodiments, a Poly-A particle is prepared with a carrier thatprotects against rapid elimination from the body. For example, acontrolled release formulation can be used, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc.

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

Additionally, suspensions of a Poly-A particle may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils, such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate, triglycerides, or liposomes. Non-lipidpolycationic amino polymers may also be used for delivery. Optionally,the suspension may also include suitable stabilizers or agents toincrease the solubility of the compounds and allow for the preparationof highly concentrated solutions.

In some embodiments, it is especially advantageous to formulate oral orparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of a Poly-Aparticle calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of Poly-A particles described herein aredictated by and directly dependent on the characteristics of theparticular Poly-A VTP and the particular therapeutic effect to beachieved, and the limitations inherent in the art of compounding such anactive agent for the treatment of individuals.

Pharmaceutical compositions comprising at least one Poly-A particle caninclude one or more pharmaceutical excipients. Examples of suchexcipients include, but are not limited to, binding agents, fillingagents, lubricating agents, suspending agents, sweeteners, flavoringagents, preservatives, buffers, wetting agents, disintegrants,effervescent agents, and other excipients. Such excipients are known inthe art. Exemplary excipients include: (1) binding agents which includevarious celluloses and cross-linked polyvinylpyrrolidone,microcrystalline cellulose, such as AVICEL. PH101 and AVICEL. PH102,silicified microcrystalline cellulose (ProSolv SMCC), gum tragacanth andgelatin; (2) filling agents such as various starches, lactose, lactosemonohydrate, and lactose anhydrous; (3) disintegrating agents such asalginic acid, Primogel, corn starch, lightly crosslinked polyvinylpyrrolidone, potato starch, maize starch, and modified starches,croscarmellose sodium, cross-povidone, sodium starch glycolate, andmixtures thereof; (4) lubricants, including agents that act on theflowability of a powder to be compressed, include magnesium stearate,colloidal silicon dioxide, such as AEROSIL 200, talc, stearic acid,calcium stearate, and silica gel; (5) glidants such as colloidal silicondioxide; (6) preservatives, such as potassium sorbate, methylparaben,propylparaben, benzoic acid and its salts, other esters ofparahydroxybenzoic acid such as butylparaben, alcohols such as ethyl orbenzyl alcohol, phenolic compounds such as phenol, or quaternarycompounds such as benzalkonium chloride; (7) diluents such aspharmaceutically acceptable inert fillers, such as microcrystallinecellulose, lactose, dibasic calcium phosphate, saccharides, and/ormixtures of any of the foregoing; examples of diluents includemicrocrystalline cellulose, such as AVICEL PH101 and AVICEL. PH102;lactose such as lactose monohydrate, lactose anhydrous, and PHARMATOSE.DCL21; dibasic calcium phosphate such as EMCOMPRESS; mannitol; starch;sorbitol; sucrose; and glucose; (8) sweetening agents, including anynatural or artificial sweetener, such as sucrose, saccharin sucrose,xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame; (9)flavoring agents, such as peppermint, methyl salicylate, orangeflavoring, MAGNASWEET (trademark of MAFCO), bubble gum flavor, fruitflavors, and the like; and (10) effervescent agents, includingeffervescent couples such as an organic acid and a carbonate orbicarbonate. Suitable organic acids include, for example, citric,tartaric, malic, fumaric, adipic, succinic, and alginic acids andanhydrides and acid salts. Suitable carbonates and bicarbonates include,for example, sodium carbonate, sodium bicarbonate, potassium carbonate,potassium bicarbonate, magnesium carbonate, sodium glycine carbonate,L-lysine carbonate, and arginine carbonate. Alternatively, only thesodium bicarbonate component of the effervescent couple may be present.

In various embodiments, the formulations described herein aresubstantially pure. As used herein, “substantially pure” means theactive ingredient (e.g., Poly-A particle) is the predominant speciespresent (i.e., on a molar basis it is more abundant than any otherindividual species in the composition). In one embodiment, asubstantially purified fraction is a composition wherein the activeingredient comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will include more than about 80% of all macromolecularspecies present in the composition. In various embodiments, asubstantially pure composition will include at least about 85%, at leastabout 90%, at least about 95%, or at least about 99% of allmacromolecular species present in the composition. In variousembodiments, the active ingredient is purified to homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

Kits Comprising Poly-A Polymer Particle Compositions

The present disclosure provides kits comprising any of the Poly-Aparticles described herein. Such kits can comprise, for example, (1) atleast one Poly-A particle; and (2) at least one pharmaceuticallyacceptable carrier, such as a solvent or solution. Additional kitcomponents can optionally include, for example: (1) any of thepharmaceutically acceptable excipients identified herein, such asstabilizers, buffers, etc., (2) at least one container, vial or similarapparatus for holding and/or mixing the kit components; and (3) deliveryapparatus.

In certain embodiments, the present invention provides instructions foradministering said inhibitors of inflammation (e.g., a Poly-A) to asubject. In certain embodiments, the present invention providesinstructions for using the compositions contained in a kit for thetreatment of conditions characterized by inflammation in a cell ortissue (e.g., providing dosing, route of administration, decision treesfor treating physicians for correlating patient-specific characteristicswith therapeutic courses of action). In certain embodiments, the presentinvention provides instructions for using the compositions contained inthe kit to treat a variety of medical conditions associated withinflammation (e.g., ALI/ARDS, sepsis, infection, arthritis, rheumatoidarthritis, juvenile rheumatoid arthritis). In certain embodiments, thepresent invention provides instructions for using the compositionscontained in the kit to treat a variety of medical conditions associatedwith inflammation, and/or autoimmune conditions.

Methods of Treatment

In some embodiments, provided herein are methods of preventing ortreating (e.g., alleviating one or more symptoms of) medical conditionsthrough the use of a Poly-A particle. The methods comprise administeringa therapeutically effective amount of a Poly-A particle to a subject inneed thereof. The described Poly-A particles can also be used forprophylactic therapy. In some embodiments, the Poly-A particle isadministered intravenously. Poly-A particles used in methods oftreatment can be a Poly-A VTP or nTP described herein, or apharmaceutically acceptable salt thereof, or a prodrug thereof. Theindividual or subject can be any animal (domestic, livestock or wild),including, but not limited to, cats, dogs, horses, pigs and cattle, andpreferably human subjects. As used herein, the terms patient,individual, and subject may be used interchangeably.

As used herein, “treating” describes the management and care of apatient for the purpose of treating a disease, condition, or disorderand includes the administration of a Poly-A particle to prevent theonset of the symptoms or complications of a disease, condition ordisorder; to alleviate symptoms or complications of the disease,condition, or disorder; or to eliminate the presence of the disease,condition or disorder in the patient. More specifically, “treating”includes reversing, attenuating, alleviating, minimizing, suppressing orhalting at least one deleterious symptom or effect of a disease(disorder) state, disease progression, disease causative agent or otherabnormal condition. Treatment is generally continued as long as symptomsand/or pathology ameliorate.

In some embodiments, compositions and methods of the present inventionare used to prevent, treat, and/or ameliorate inflammatory diseases,malignant diseases, infections, autoimmune diseases, and/or otherdiseases or conditions in which neutrophil pathophysiology isimplicated. Non-limiting exemplary inflammatory diseases that may betreated with the Poly-A particles described herein include rheumatoidarthritis, juvenile idiopathic arthritis, systemic-onset juvenileidiopathic arthritis, gout, osteoarthritis, sepsis, asthma, interstitiallung disease, inflammatory bowel disease, systemic sclerosis,intraocular inflammation, Grave's disease, endometriosis, systemicsclerosis, adult-onset still disease, amyloid A amyloidosis, polymyalgiarheumatic, remitting seronegative symmetrical synovitis with pittingedema, Behcet's disease, uveitis, graft-versus-host diseases, andTNFR-associated periodic syndrome. Malignant diseases that may betreated with the Poly-A particles described herein include cancers andcancer-related conditions. Non-limiting exemplary cancers includemultiple myeloma, leukemia, pancreatic cancer, breast cancer, colorectalcancer, cachexia, melanoma, cervical cancer, ovarian cancer, lymphoma,gastrointestinal, lung cancer, prostate cancer, renal cell carcinoma,metastatic kidney cancer, solid tumors, non-small cell lung carcinoma,non-Hodgkin's lymphoma, bladder cancer, oral cancer, myeloproliferativeneoplasm, B-cell lymphoproliferative disease, and plasma cell leukemia.Non-limiting exemplary cancer-related conditions include non-small celllung cancer-related fatigue and cancer related anorexia. Non-limitingexemplary infections that may be treated with the Poly-A particlesdescribed herein include bacterial infections, viral infections, fungalinfections, rickettsial infections, parasitic infections, humanimmunodeficiency virus (HIV) infections, human T-lymphotropic virus(HTLV) infections, respiratory virus infections, cerebral malaria,urinary tract infections, and meningococcal infections. Non-limitingexemplary autoimmune diseases that may be treated with the Poly-A VTPsdescribed herein include systemic lupus erythromatosus, systemicsclerosis, polymyositis, vasculitis syndrome including giant cellarteritis, Takayasu aeteritis, cryoglobulinemia,myeloperoxidase-antineutrophil cytoplasmic antibody-associatedcrescentic glomerulonephritis, rheumatoid vasculitis, Crohn's disease,relapsing polychondritis, acquired hemophilia A, and autoimmunehemolytic anemia. Further diseases that may be treated with the Poly-Aparticles described herein include, but are not limited to, Castleman'sdisease, ankylosing spondylytis, coronary heart disease, cardiovasculardisease in rheumatoid arthritis, pulmonary arterial hypertension,chronic obstructive pulmonary disease (COPD), atopic dermatitis,psoriasis, sciatica, type II diabetes, obesity, giant cell arteritis,acute graft-versus-host disease (GVHD), non-ST elevation myocardialinfarction, anti-neutrophil cytoplasmic antibody (ANCA) associatedvasculitis, neuromyelitis optica, chronic glomerulonephritis, andTakayasu arteritis.

In some embodiments, the disclosed compounds or pharmaceuticallyacceptable salts thereof, or prodrugs, can be administered incombination with other active agents. Compositions including thedisclosed Poly-A particles may contain, for example, more than onePoly-A particle. In some embodiments, a composition containing one ormore Poly-A particles are administered in combination with one or moreadditional agents for preventing, treating, and/or amelioratinginflammatory diseases, malignant diseases, infections, autoimmunediseases, and/or other diseases or conditions in which neutrophilpathophysiology is implicated.

The dosage regimen utilizing the Poly-A particles is selected inaccordance with a variety of factors, including, for example, type,species, age, weight, sex and medical condition of the subject; theseverity of the condition to be treated; the route of administration;the renal and hepatic function of the subject; and the particular Poly-Aparticle or carrier thereof employed. An ordinarily skilled physician orveterinarian can readily determine and prescribe the effective amount ofthe composition required to prevent, counter or arrest the progress ofthe condition. In general, the dosage, i.e., the therapeuticallyeffective amount, ranges from about 1 ng/kg to about 1 g/kg body weight,in some embodiments about 1 ug/kg to about 1 g/kg body weight, in someembodiments about 1 ug/kg to about 100 mg/kg body weight, in someembodiments about 1 ug/kg to about 10 mg/kg body weight of the subjectbeing treated, per day.

Methods for Diagnosing and Detecting

Poly-A particles, described herein, find use as diagnostic reagents,either in vitro or in vivo. The Poly-A particles identified herein canbe used in any diagnostic, detection, imaging, high throughput screeningor target validation techniques or procedures or assays for which Poly-Aparticles without limitation can be used.

Poly-A particles capable of binding restraining neutrophils, describedherein, find use in a variety of assays including, assays that useplanar arrays, beads, and other types of solid supports. The assays maybe used in a variety of contexts including in life science researchapplications, clinical diagnostic applications, (e.g., a diagnostic testfor a disease, or a “wellness” test for preventative healthcare),affinity-linked oligonucleotide nuclease assay (ALONA) andubiquitin-proteasome system (UPS) assays, and in vivo imagingapplications. For some applications, multiplexed assays employing thedescribed Poly-A VTPs and may be used.

In some embodiments, the Poly-A particles are used as sensitive andspecific reagents for incorporation into a variety of in vitrodiagnostic methods or kits. In some embodiments, the Poly-A particlesare used as substitutes for antibodies in a number of infectious, orother type of disease detection methods where the Poly-A particleincludes either or both a detectable material and an immobilization orcapture component. In these embodiments, after the Poly-A particle fromthe kit is mixed with a clinical specimen, a variety of assay formatsmay be utilized. In one embodiment, the Poly-A particles also include adetectable label, such as a fluorophore. In other embodiments, the assayformat may include fluorescence quenching, hybridization methods, flowcytometry, mass spectroscopy, inhibition or competition methods, enzymelinked oligonucleotide assays, SPR, evanescent wave methods, etc. Insome embodiments, the Poly-A particle is provided in the kit insolution. In other embodiments, the Poly-A particle in the kit isimmobilized onto a solid support used in conjunction with the assay fortesting the specimen. In various embodiments, the solid support isdesigned for the detection of one or more targets of interest. In otherembodiments, the kit may further include reagents to extract the targetof interest, reagents for constructing Poly-A particles, reagents forperforming washing, detection reagents, etc.

Diagnostic or assay devices, e.g. columns, test strips or biochips,having one or more Poly-A particles adhered to a solid surface of thedevice are also provided. The Poly-A particles may be positioned so asto be capable of binding neutrophils that are contacted with the solidsurface to form Poly-A particle-neutrophil complex that remains adheredto the surface of the device, thereby capturing the target and enablingdetection and optionally quantitation of the target. An array of Poly-Aparticles (which may be the same or different) may be provided on such adevice.

In one embodiment for detecting neutrophils, a Poly-A particle iscontacted with a labeling agent that includes a binding partner that isspecific for a neutrophil. The specific binding partner may be anysuitable moiety, including an antibody, an antibody fragment, asynthetic antibody mimetic, a biomimetic, an aptamer, a molecularimprinted ligand, and the like. The specific binding partner isconjugated or linked to another labeling agent component, usually, adetectable moiety or label.

The detectable moiety or label is capable of being detected directly orindirectly. In general, any reporter molecule that is detectable can bea label. Labels include, for example, (i) reporter molecules that can bedetected directly by virtue of generating a signal, (ii) specificbinding pair members that may be detected indirectly by subsequentbinding to a cognate that contains a reporter molecule, (iii) mass tagsdetectable by mass spectrometry, (iv) oligonucleotide primers that canprovide a template for amplification or ligation, and (v) a specificpolynucleotide sequence or recognition sequence that can act as aligand, such as, for example, a repressor protein, wherein in the lattertwo instances the oligonucleotide primer or repressor protein will have,or be capable of having, a reporter molecule, and so forth. The reportermolecule can be a catalyst, such as an enzyme, a polynucleotide codingfor a catalyst, promoter, dye, fluorescent molecule, quantum dot,chemiluminescent molecule, coenzyme, enzyme substrate, radioactivegroup, a small organic molecule, amplifiable polynucleotide sequence, aparticle such as latex or carbon particle, metal sol, crystallite,liposome, cell, etc., which may or may not be further labeled with adye, catalyst or other detectable group, a mass tag that alters theweight of the molecule to which it is conjugated for mass spectrometrypurposes, and the like. The label can be selected from electromagneticor electrochemical materials. In one embodiment, the detectable label isa fluorescent dye. Other labels and labeling schemes will be evident toone skilled in the art based on the disclosure herein.

EXAMPLES

The invention, now being generally described, will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.All protocols for animal and human studies were approved by theUniversity of Michigan's Committee on Use and Care of Animal or theInstitutional Review Board.

Example 1—Poly-A Particles Reduce Neutrophil Lung Migration in ALI

Poly-A microspheres were injected into the tail vein of mice withLPS-induced ALI at 30 mg/kg. Poly-A spheres injected 1 hr after LPSadministration significantly reduced the BALF neutrophil count comparedto ALI mice with no particles (FIG. 3). Tail vein injection of aspirinin DMSO did not reduce the BALF neutrophil count in ALI mice,demonstrating the utility of the Poly-A VTP particle-neutrophil physicalinteraction in the observed reduction of neutrophil lung migration inALI. Particle-treated ALI mice had lower albumin levels in the BALF(FIG. 16), denoting a reduction of lung injury with particle injection.The levels for markers of inflammation typically found in the BALF inALI mice, showed that the Poly-A spheres reduce inflammation in lungsignificantly (p<0.05) compared to PS or aspirin. BALF concentration ofTNF-α, KC (FIG. 15), and MIP2 (FIG. 16) were reduced in Poly-A-treatedALI-mice, but not for mice treated with PS spheres or aspirin.

Example 2—Poly-A MPs do not Exacerbate Bacterial Infection in Mice withBacteria-Induced ALI

To determine whether Poly-A MPs have utility in the clinical setting ofALI/ARDS, i.e., whether Poly-A MP therapy functions without affecting ahost's capacity to clear a primary infection, a mouse model of ALI/ARDSinfected with Pseudomonas (P.) aeruginosa was investigated. Mice wereinfected with P. aeruginosa via intratracheal (i.t.) inoculation. P.aeruginosa bacteria grown in Difco broth were diluted to the desiredconcentration. Mice were anesthetized with ketamine and xylazine by theintraperitoneal route, the trachea exposed, and a 30 μl inoculum(7-8×10⁵ CFU), or saline, were administered via a sterile 26-gaugeneedle. At either 6 hrs or 18 hrs after infection, Poly-A MPs wereintravenously injected (IV; tail vein), and mice were euthanized at 24hrs after infection. At 24 hrs after infection, the blood CFU wassignificantly lower in the Poly-A MP treated mice than in the untreatedmice for both the 6 hrs and 18 hrs injection points (FIG. 4). Results inFIG. 5 show that the injection of Poly-A MPs at the 18 hrs intervalreduced neutrophils in the lung BALF at the 24 hr sample collection.Conversely, the neutrophil count in the BALF for the Poly-A MP injectionat the 6 hr interval was the same as for non-treated, infected mice.These data show that Poly-A MPs do not negatively impact a host'sability to clear a primary lung infection. In keeping with BALFneutrophil data, Poly-A MP treatment at the 18 hrs interval prevents thesystemic dissemination of the infection, with no trace of P. aeruginosafound in blood at 24 hrs after infection. Lower concentrations of TNF-α(FIG. 4) IL-6, KC, and MCP-1 in the BALF with Poly-A MP treatment (FIG.16) were observed Taken together, these data indicate that the timing ofparticle injection is important for efficacy in treating ALI. In view ofthe lower 24 hrs BALF neutrophil count for particle treatment at 18 hrsafter infection vs. treatment at 6 hrs, a ramp-up interval afterbacterial infection but before particles are injected may underly atherapeutic effect. Although the present invention is not confined to aspecific mechanism or mechanisms, a minimum number of neutrophils mayfirst enter lung tissue to facilitate bacteria clearance, but beforeextensive damage is done to the lungs. Thereafter, Poly-A MPs haltneutrophil lung transmigration, and mitigate injury while allowingnatural host immunity or an active pharmaceutic ingredient (API) toclear the primary infection.

Example 3—Neutrophil-Poly-A MP Interactions in LPS-Induced Lung InjuryModels Methods

To determine whether Poly-A MP administration mitigates neutrophilinfiltration into the lungs in a murine model of ALI, and to determinewhether Poly-A MP particles have therapeutic advantages compared topolystyrene (PS) particles and vehicle controls, an LPS-induced model ofALI was used, and the total BALF cells, % of BALF neutrophils andmacrophages, and inflammatory cytokine concentrations were quantifiedand compared.

Particle Degradation

1×10⁷ Poly-A nTP particles as determined by manual count on ahemocytometer were suspended in 10 mL of PBS −/− and placed underrotation at 37° C. (FIG. 10) The particles were centrifuged and thesupernatant was serially removed and replaced. The supernatant was thenadded to a 96-well plate and the fluorescence intensity was measured(ex=315 nm and em=408 nm for salicylic acid). The cumulativefluorescence intensity at each timepoint is plotted versus degradationtime in FIG. 12 (MFI=mean fluorescent intensity)

Acute Lung Injury (ALI) Model

Male BALB/c or C57BL/6J mice were anesthetized with inhaled isoflurane,and given 50 uL of 0.4 mg/mL LPS orotracheally to induce lunginflammation. Particles (2×10⁸ in 100 uL PBS per mouse) were injected ateither 2 hrs or 6 hrs post-instillation via tail vein catheter. Methodsof euthanasia and sample processing are shared with the P. aeruginosamodel (below) except instead of cytospin BALF, cells were stained withCD45, CD11b, and Ly6G, and were fixed overnight before flow cytometry todetermine the % of neutrophils and macrophages in each sample.

Results

Poly-A nTP administration reduces neutrophil infiltration and theconcentration of inflammatory cytokines in the lungs after LPS, denotingmitigation of neutrophil mediated inflammatory responses. (FIGS. 14, 15,and 16.) Both PS and Poly-A nTPs reduce neutrophil infiltration into thelungs after LPS, but only Poly-A MP reduces the concentration ofinflammatory cytokines in the lungs. (FIGS. 17, 18, 19, 20, 21, and 22.)

Example 4—Optimal Administration Intervals

To characterize the timeline of neutrophil influx into the lungs, and toidentify the optimal injection interval after infection for maximumtherapeutic benefit, a model of intratracheal LPS administration inC57BL/6J mice was used for analysis of BALF samples. (FIG. 23). Influxof neutrophils into the alveolar space is greatest between 2 hrs and 6hrs post-instillation. A 2 hr post-instillation Poly-A MP administrationinterval confers maximum reduction in neutrophil infiltration in animalsunder these conditions. (FIGS. 24, 25, 26, 27, 28 and 29.) (Neutrophil NΦ. and Macrophage M Φ.)

Example 5—Therapeutic Benefit of Poly-A MP Administration

The therapeutic benefits of Poly-A MP administration were identifiedusing a bacterial model of ALI/ARDS in which mice were infected with P.aeruginosa, and Poly-A MPs were parenterally administered at 6 and 18hours after infection.

Methods Particle Preparation

Poly-A particles were prepared using a single emulsion solventevaporation (ESE) method. 20 mg of Poly-A polymer were dissolved in 20mL of dichloromethane. Then, 75 mL of 1% polyvinyl alcohol (PVA)solution was placed on a mixer at 4250 rpm. Poly-A solution was slowlyinjected into the PVA solution, and allowed to mix for 2 hrs. Aftermixing, the solution was allowed to settle for ˜45 minutes, and theparticles were removed and washed by centrifugation 3× with DI water.Particles were then lyophilized and stored at −40° C. until use.Particle size was determined by scanning electron microscopy (SEM) anddynamic light scattering (DLS) and particle concentration was determinedvia counting on a hemocytometer.

Bacterial Growth

P. aeruginosa cultures were grown overnight in Difco nutrient broth at37° C. under constant shaking. The concentration of bacteria in thebroth was determined by measuring light absorbance at 600 nm, thenplotting the optical density (OD) on a standard curve generated by knowncolony forming units (CFUs)

Intranasal Bacterial Inoculation

Mice were anesthetized with an intraperitoneal injection ofketamine/xylazine mixture, and given 30 uL of bacteria solution (15 uLin each nostril) intranasally to induce lung infection. (FIG. 30.)

Poly-A VTP Injection

At 6 hrs or 19 hrs post-infection, mice were placed in a restrainer, anda catheter was inserted into the tail vein. Each mouse received 2×10⁸particles in 100 uL of injection volume, for a dose of approximately 30mg/kg. (FIG. 30.)

Euthanasia and Sample Processing

24 hours post-infection, mice were euthanized via CO2 overdose. Aftereuthanasia, the chest cavity was exposed and a cardiac puncture was usedto collect blood. The trachea was exposed and opened, and the lungs werelavaged with 3 mL of PBS −/− to remove cells in the alveolar space. BALFsamples were centrifuged and supernatants were stored at −80° C. forELISA to quantify inflammatory cytokines. The cell pellets wereresuspended in 500 uL of RPMI media, and aliquots were diluted 1:1 withTurk Blood Diluting Fluid and counted via hemacytometer. Cytospinsamples were prepared and cells were stained to differentiateneutrophils from mononuclear cells. Blood and BALF samples were platedon agar plates and allowed to grow overnight at 37° C. to determineCFUs. Blood was centrifuged and plasma samples were collected and storedat −80° C. for ELISA analysis.

Results

Poly-A MP administration at 6 hrs and 18 hrs reduced lung concentrationsof IL-6, IL-10, KC, MIP2, MCP1, and TNF-α cytokines relative to P.aeruginosa infected but untreated control mice, indicating reducedpulmonary inflammation after Poly-A VTP treatment. (FIGS. 31, 32, and33.) Poly-A MP treatment at 18 hrs after infection significantly reducedthe number of neutrophils in BALF, but administration 6 hrs afterinfection did not. (FIG. 34.) Poly-A MP treatment at 18 hrs afterinfection reduced bacterial CFU in blood indicating that treatmentserves to confine infection to the lungs.

Example 6—Therapeutic Benefit of Poly-A MP Administration

To ensure that Poly-A MPs cause a reduction in bacterial CFU bloodcounts, the Methods of Example 5 were repeated with 18 hr Poly-A MPparticle administration in parallel with a saline intravenous (IV)control. Poly-A MP administration, but not saline, reduced CFU in theblood relative to the infected controls, indicating that Poly-A MPscontain infection to the lungs, and prevent systemic spread of theinfection.

Example 7—Time Course of Pulmonary Neutrophil Infiltration afterInfection

To determine the time course of neutrophil influx into the lungs afterinfection BALF cell counts, BALF P. aeruginosa 19660 counts, and bloodP. aeruginosa 19660 counts at 12 hrs, 18 hrs, 24 hrs, 30 hrs and 36 hrsafter infection were measured. A marked increase in BALF cells wasobserved between 12 and 24 hours, with an associated increase in bothlung and blood P. aeruginosa CFU counts. (FIGS. 35 and 36.)

Example 8—Therapeutic Benefit of Poly-A MP on Survival after Infection

To determine whether Poly-A MP particle administration has a positiveimpact on survival post-infection, mice were infected with P.aeruginosa, and half the mice were given Poly-A particles 18 hourspost-infection. Poly-A MPs significantly reduced mortality in theinfected mice. (FIG. 37.)

Example 9—Poly-A Particle Conjugation

To confirm that the Poly-A particles comprise sufficient carboxyl groupson the surface with which to conjugate targeting ligands,1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC)chemistry was used to conjugate Neutravidin followed by biotinylatedanti-ICAM-1 bound to the surface of the particles. 2 μm Poly-A particleswere suspended in 50 mM MES buffer and counted using a hemacytometer.2.2×10⁹ particles were suspended in 400 uL of 5 mg/mL Neutravidinsolution in 50 mM MES. Particles were rotated for 15 minutes, afterwhich 400 uL of 75 mg/mL EDC solution was added, and particles wererotated overnight. Then, 4 mg of glycine was added to the particlesolution to quench the reaction, and particles were rotated for 15minutes. Particles were then centrifuged and resuspended in PBS −/−.After Neutravidin conjugation, 1×10⁷ particles were incubated in 100 uLof 5 mg/mL biotinylated anti-ICAM-1 in PBS −/− with 2% BSA for 1 hour onrotation. Avidin particles were then stained with Biotin-PE andanti-ICAM particles were stained with goat anti-mouse IgG-fluoresceinisothiocyanate (FITC). Particles were then measured on an Attune FlowCytometer, and compared to R-phycoerythrin (R-PE) and FITC molecules ofequivalent soluble fluorochrome (MESF) beads to determine the totalnumber of ligand sites on the particle surface. FIG. 38 shows flowcytometry histogram plots. Poly-A-Avidin and Poly-A-anti-ICAM had 36,819sites/um² and 43,235 sites/um², respectively.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

INCORPORATION BY REFERENCE

All publications, published patent documents, and patent applicationscited herein are hereby incorporated by reference to the same extent asthough each individual publication, published patent document, or patentapplication was specifically and individually indicated as beingincorporated by reference.

1.-14. (canceled)
 15. A kit comprising a pharmaceutical composition,comprising a Poly-A particle, and instructions for administering saidpharmaceutical composition to a patient diagnosed with vascularthrombosis, inflammatory arthritis, systemic lupus erythematosus (SLE),atherosclerosis, sepsis, acute lung injury arthritis (ALI) and acuterespiratory distress syndrome (ARDS).
 16. A pharmaceutical compositioncomprising at least one Poly-A particle and a pharmaceuticallyacceptable carrier, and/or a pharmaceutically acceptable formulation.17. The pharmaceutical composition of claim 16, wherein said at leastone Poly-A particle is made by the method of: a) dissolving polyvinylalcohol (PVA) with an average molecular weight of 20-70 kDA in water togenerate a 1 wt % PVA solution of pH 6-7; b) dissolving Poly-A indichloromethane (DCM); c) adding said Poly-A in said DCM to said PVAsolution over at least one hour during mixing at >4000 rpm to generatean emulsion; d) centrifuging said emulsion; e) aspirating a centrifugedsolution from a centrifuged pellet; f) resuspending said pellet indeionized water to generate suspended Poly-A particles; g) washing saidsuspended Poly-A particles; h) lyophilizing said washed Poly-Aparticles; and i) freezing said lyophilized Poly-A particles.
 18. Thepharmaceutical composition of claim 17, wherein said Poly-A particle ismodified to be a carrier of one or more hydrophobic bioactive compoundsor drugs by adding said one or more hydrophobic bioactive compounds ordrugs to said Poly-A polymer dissolved in said DCM.
 19. Thepharmaceutical composition of claim 17, wherein said Poly-A particle ismodified to be a carrier of one or more hydrophilic bioactive compoundsor drugs by adding said one or more of said hydrophilic bioactivecompounds or drugs to a water phase that is emulsified into said Poly-Apolymer dissolved in said DCM, and emulsifying said drug-polymeremulsion in a solution of 1 wt % PVA in water.
 20. The pharmaceuticalcomposition of claim 16, wherein said Poly-A particle is a targetedPoly-A particle.
 21. The pharmaceutical composition of claim 20, whereinsaid targeted Poly-A particle is a Poly-A VTP particle.
 22. Thepharmaceutical composition of claim 21, wherein said Poly-A VTP particleis made by the method of: a) suspending Poly-A particles in 50 mM IVIESbuffer; b) suspending said particles in Neutravidin solution in 50 mMIVIES; c) adding 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimidehydrochloride (EDC) solution to said suspended particles; d) addingglycerine to said solution comprising said Poly-A particles; e)centrifuging said solution; f) resuspending said Poly-A particles inPBS; and j) incubating said solution comprising said Poly-A particleswith biotinylated anti-ICAM-1 in PBS −/− with 2% BSA.
 23. Thepharmaceutical composition of claim 16, wherein said Poly-A particle isa Poly-A nTP particle.